title: Probing the stability of the "naked" mucin-like domain of human α-dystroglycan.
abstract: BACKGROUND
  α-Dystroglycan (α-DG) is heavily glycosylated within its central mucin-like domain. The glycosylation shell of α-dystroglycan is known to largely influence its functional properties toward extracellular ligands. The structural features of this α-dystroglycan domain have been poorly studied so far. For the first time, we have attempted a recombinant expression approach in E. coli cells, in order to analyze by biochemical and biophysical techniques this important domain of the α-dystroglycan core protein.
  
  
  RESULTS
  We expressed the recombinant mucin-like domain of human α-dystroglycan in E. coli cells, and purified it as a soluble peptide of 174 aa. A cleavage event, that progressively emerges under repeated cycles of freeze/thaw, occurs at the carboxy side of Arg461, liberating a 151 aa fragment as revealed by mass spectrometry analysis. The mucin-like peptide lacks any particular fold, as confirmed by its hydrodynamic properties and its fluorescence behavior under guanidine hydrochloride denaturation. Dynamic light scattering has been used to demonstrate that this mucin-like peptide is arranged in a conformation that is prone to aggregation at room temperature, with a melting temperature of ~40°C, which indicates a pronounced instability. Such a conclusion has been corroborated by trypsin limited proteolysis, upon which the protein has been fully degraded in less than 60 min.
  
  
  CONCLUSIONS
  Our analysis indirectly confirms the idea that the mucin-like domain of α-dystroglycan needs to be extensively glycosylated in order to reach a stable conformation. The absence/reduction of glycosylation by itself may greatly reduce the stability of the dystroglycan complex. Although an altered pattern of α-dystroglycan O-mannosylation, that is not significantly changing its overall glycosylation fraction, represents the primary molecular clue behind currently known dystroglycanopathies, it cannot be ruled out that still unidentified forms of αDG-related dystrophy might originate by a more substantial reduction of α-dystroglycan glycosylation and by its consequent destabilization.
authors.forename: Manuela
authors.initials: M
authors.lastname: Bozzi
authors.forename: Enrico
authors.initials: E
authors.lastname: Di Stasio
authors.forename: Giovanni Luca
authors.initials: GL
authors.lastname: Scaglione
authors.forename: Claudia
authors.initials: C
authors.lastname: Desiderio
authors.forename: Claudia
authors.initials: C
authors.lastname: Martelli
authors.forename: Bruno
authors.initials: B
authors.lastname: Giardina
authors.forename: Francesca
authors.initials: F
authors.lastname: Sciandra
authors.forename: Andrea
authors.initials: A
authors.lastname: Brancaccio
mesh_terms: D049030:Dystroglycans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008027:Light; D013058:Mass Spectrometry; D011489:Protein Denaturation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012542:Scattering, Radiation
chemical_list: C483081:DAG1 protein, human; D011994:Recombinant Proteins; D049030:Dystroglycans